Seventy- two hours after plating, the cells were washed and resuspended in serum and azide free PBS (Life Technologies, Carlsbad, CA) and were stained with the eFluor 780 Viability dye (eBioscience, San Diego, CA) for 30 minutes at 4°C. Following washing, the cells were incubated with anti-mouse CD4-FITC (RM4-5) and CD25-APC (PC61.5) or with anti-human CD4-FITC (OKT4) (eBioscience, San Diego, CA) and CD25-PE (BD Biosciences, San Jose, CA) for 30 minutes at room temperature. A labeled isotype-matched control antibody was used as a control in these experiments. Cells were fixed and permeabilized using the FoxP3 Staining Buffer Set (eBioscience, San Diego, CA), and then stained with anti-mouse FoxP3-PE (FJK-16s), anti-human FoxP3-APC (236A/E7) (eBioscience, San Diego, CA) or anti-human LAP-PerCP-Cy5.5 (BD Biosciences, San Jose, CA). Human samples were gated for CD4 expression prior to gating for FoxP3 expression. Samples were run on a benchtop BD FACSCanto flow cytometer (BD Biosciences, San Diego, CA) and analysis was performed using FlowJo FACS analysis software (FlowJo LLC, Ashland, OR).
Facs analysis software
Manufactured by FlowJo
Sourced in United States
FlowJo is a data analysis software designed for flow cytometry. It provides tools for visualizing and analyzing data generated from flow cytometry experiments. The software allows users to import, process, and analyze flow cytometry data files.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (2 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (2 mentions)
Facscan, by BD (2 mentions)
Viability dye efluor 780, by Thermo Fisher Scientific (1 mentions)
Cd25 pe, by BD (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Anti flag fitc antibody, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Facscanto, by BD (1 mentions)
Cellquest pro, by BD (1 mentions)
7 aminoactinomycin d 7 aad, by BD (1 mentions)
Rituxan, by Genentech (1 mentions)
Cell quest data acquisition software, by BD (1 mentions)
Fitc anti human igg fc, by BioLegend (1 mentions)
5 protocols using facs analysis software
1
Quantifying Regulatory T-cell Markers
2
Quantifying Apoptosis in Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following treatment with 3 µM BaA, BaP or BEP for 72 h, cell apoptosis was determined by staining mLFCs with propidium iodide (PI) and Annexin V-fluorescein isothiocyanate (FITC). Cells (1×106) were washed with PBS and centrifuged at 200 × g for 5 min at room temperature. The cellular pellet was suspended in 50 µl Annexin V solution containing 5 µl Annexin V-FITC and 5 µl PI for 15 min at room temperature, provided in the Annexin V-FITC Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA). Data acquisition and data analysis were performed using a flow cytometer (FACScan; Becton-Dickinson and Company, Franklin Lakes, NJ, USA) with the FlowJo FACS analysis software (version 10.0; FlowJo LLC, Ashland, OR, USA).
3
Immunophenotyping of Engineered T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed and resuspended in serum free and azide free PBS (Life Technologies) and were stained with A2AR-FITC, CD4-FITC or CD25-PE for 30 minutes at room temperature. Cells transduced with lentiCRISPR loxP A2AR virus were marked using anti-FLAG-FITC antibody (Sigma-Aldrich, St. Louis, MO, USA) after fixing and permeabilization. An isotype control was used for each fluorophore used. For FoxP3 staining, cells were fixed and permeabilized for 30 minutes at room temperature using the eBioscience FoxP3 staining buffer set (Affymetrix, San Diego, CA, USA), followed by staining with anti-human FoxP3-APC (Affymetrix). Samples were run on a benchtop BD FACScanto flow cytometer (BD Biosciences, San Diego, CA, USA) and a FACScan (BD Biosciences). Analysis was performed using FlowJo FACS analysis software (FlowJo LLC, Ashland, OR, USA) and CellQuestPro (BD Biosciences).
4
CD20 Expression on Lymphoma Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The percentage of CD20 antigen present on cultured Wil2-S and Daudi lymphoma cells was assessed by FACS Calibur flow cytometry (BD Biosciences, San Jose, CA, USA). Daudi and Wil2-S cells at 1 × 106 cells/mL were labeled with FITC Mouse Anti-Human CD20 (BD Biosciences, San Jose, CA, USA). After incubating 100 µL of cells with 5 µL of FITC Mouse Anti-Human CD20 for 30 min at 4 °C, cells were washed and resuspended in stain buffer (Phosphate Buffered Saline (PBS) containing 2% fetal bovine serum) for flow cytometry analysis. The nucleic acid dye 7-Amino-Actinomycin D (7-AAD) (BD Biosciences, San Jose, CA, USA) was used as 0.5 µL/500 µL for the exclusion of nonviable cells in flow cytometry analysis. The relative FITC fluorescence was determined with 488 nm excitation, and emission detected with 530/30 nm using a 530/30 bandpass filter. Percentage of cells with specific CD20-FITC staining was determined with FACS plots using FlowJo FACS analysis software (FlowJo, Ashland, OR, USA).
5
Comparative Binding Analysis of Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flow cytometry analysis was performed to determine and compare the binding of NbRTXA2G2 and Rituxan® (Genentech, South San Francisco, CA, USA) antibodies to target cells (Wil2-S and Daudi) using a FACS Calibur (BD Biosciences, San Jose, CA, USA). Briefly, 100 µL cells at 1 × 106 cells/mL were incubated with different concentrations of Rituxan® standard and NbRTXA2G2 for 30 min at 4 °C. Cells were then washed and incubated with 5 µL of FITC anti-human IgG Fc (BioLegend, San Diego, CA, USA). FITC Mouse IgG2a, k Isotype Ctrl (FC) (BioLegend, San Diego, CA, USA) was used as isotype control. Cells were washed with stain buffer and analyzed by flow cytometry. Nucleic acid dye 7-AAD was used for the quantification of dead cells in each sample as 25 ng/500 µL. Cell Quest data acquisition software (BD Biosciences, San Jose, CA, USA) and Flowjo FACS analysis software (FlowJo, Ashland, OR, USA) were used to derive data plots.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!